Sequence-specific DNA amplification has many applications in molecular biology research and medical diagnostics. At the present time, there are two main strategies for amplifying a defined sequence of nucleic acid: polymerase chain reaction (PCR) and isothermal amplification. The polymerase chain reaction relies upon instrument-based thermal cycling to denature template DNA, followed by the annealing of primers at specific sites in the denatured template and extension of the primers by a thermostable DNA polymerase (such as Taq polymerase) in order to exponentially increase the amount of DNA [U.S. Pat. Nos. 4,683,202 and 4,683,195].
Isothermal amplification of DNA may require the same three steps and an initial high temperature incubation to denature the template DNA for initiation of the process, but the amplification of the DNA to take place at one defined temperature.
A variety of isothermal amplification methods have been developed, for example: strand displacement amplification (SDA) [Walker, G. T. et al. Nucleic Acids Res 20, 1691-6 (1992); and Walker, G. T., Little, M. C., Nadeau, J. G. & Shank, D. D. Proc Natl Acad Sci USA 89, 392-6 (1992)], rolling circle amplification [Fire, A. & Xu, S. Q. Proc Natl Acad Sci USA 92, 4641-5 (1995)], cross priming amplification (CPA) [Xu, G. et al. Sci. Rep. 2, 246; D01:10.1038/srep00246 (2012)] and loop mediated amplification [Notomi, T. et al. Nucleic Acids Res 28, e63 (2000)].
These methods, like many other isothermal amplification methods, require the use of a DNA polymerase with a strong strand displacement activity. At the same time, sequence-specific DNA synthesis or amplification needs a high temperature of the reaction (as a rule 60° C. or over) for the specific annealing of primers. Thus, a DNA polymerase suitable for these methods must be a thermostable DNA polymerase with a strong strand displacement activity.
At present time, only the large fragment of Bst DNA polymerase is suitable for methods of sequence-specific isothermal DNA amplification, such as LAMP or CPA. A relative thermostability and strong strand displacement activity of Bst DNA polymerase make it helpful for other applications. For example, usage of Bst in Illumina's Next Generation Sequencing technology for carrying out cluster amplification of DNA molecules can be mentioned.
The Bst DNA polymerase or Bacillus stearothermophilus DNA Polymerase I is a typical member of polymerase family A and its structure looks like structure of Taq DNA polymerase or other members of the family [Kiefer, J. R. et al. (1997) Structure, 5, 95-108] but a mechanism of strand displacement of Bst and other A-family polymerases is unclear. Bst polymerase is one of the most popular enzymes with strand displacement activity because its optimum is at about 63° C. and it is suitable for sequence-specific amplification like LAMP. Unfortunately, Bst polymerase can be used at temperatures only up to 68-70° C. At temperature 68° C. or higher it is inactivated [Xu, G. et al. Sci. Rep. 2, 246; D01:10.1038/srep00246 (2012)]. So, Bst cannot be used in methods requiring heat denaturation of DNA, such as PCR or polymerase chain displacement reaction (PCDR) [Harris, C. L. et al. BioTechniques 54:93-97 (February 2013) doi 10.2144/000113951].
Additionally, initial heat denaturation of DNA can many times increase the sensitivity of isothermal DNA amplification like LAMP [Aryan, E., et al. (2010) Microbiol. Res., 165, 211-220; Geojith G., et al. (2010) J Microbiol Methods, 84, 71-73; Neonakis, I. K., et al. (2011) Eur. J. Clin. Microbiol. Infect. Dis., 30, 937-942].
Therefore the availability of novel DNA polymerases with high thermo stability and a strong strand displacement activity would be highly advantageous.